Several types of cancer metastasize to bone, resulting in extensive osteolysis. This in turn results in considerable morbidity including skeleta] disfunction, severe pain and reduced quality of life. This seems to stem from the ability of cancer cells to produce factors that upregulate levels of the ligand for receptor of activated NFKappaB (RANKL). RANKL induces osteoclast differentiation from macrophage cell precursor. RANKL-induced osteoclasts mediate bone resorption. Therefore, RANKL is a key factor in cancer cell-mediated metastasis to bone and osteolysis. Under physiological conditions, bone resorption is downregulated by a soluble decoy receptor, osteoprotegerin (OPG). By binding to RANKL, OPG prevents its interaction with osteoclast precursors. Intriguingly, cancer cells also produce factors that silence osteoprotegerin expression, further enhancing bone metastasis and destruction. In fact, administration of drugs that restore OPG production or OPG itself can decrease both bone destruction and skeletal tumor burden. Therefore, understanding how OPG interacts with RANKL will be of great value for the discovery of compounds that mimmick OPG in their inactivation of RANKL. Such molecules will provide a novel avenue for therapeutic intervention in metastasis to the skeleton and the extreme pain and complications resulting from this. The interaction of OPG with RANKL will be studied in order to discover the primary sequence elements determining the interaction between these two proteins. Our hypothesis is that the OPG-RANKL interaction recapitulates the essential features driving the interaction between tumor necrosis factor-alpha (TNF) and its receptor (TNFR), because OPG is a member TNF receptor superfamily and RANKL is a member of the TNF superfamily. Specifically, we will study the stoichiometry and thermodynamics of binding of OPG to RANKL by using several, independent biophysical approaches. We will also study the effect of mutations at selected amino acid residues of OPG and RANKL on the thermodynamics of OPG-RANKL complex formation in order to identify the key residues driving this interaction. Finally, we will solve the 3-dimensional structure of the OPG-RANKL complex by X-ray crystallography in order to provide a structural model for the thermodynamic and genetic information.